P489: THE LONG NON-CODING RNA LINC01547 PROMOTES PROLIFERATION, CLONOGENICITY, AND CELL CYCLE PROGRESSION IN ACUTE MYELOID LEUKEMIA
Authors: S. M. N. Zimmermann, M. F. Blank, D. Heid, M. Bruckmann, C. Müller-Tidow, J. Krijgsveld
Journal: HemaSphere (2022)

Background: Non-coding sequences account for about 98% of the human genome, and it has become increasingly apparent that non-protein coding transcripts drive numerous physiological and pathological cellular processes. Long non-coding RNAs play important roles in cancer, but functions in leukemogenesis and leukemic maintenance, as well as underlying mechanisms, remain to be elucidated. Aims: In this study, we characterize the functional and mechanistic implications of the long non-coding RNA LINC01547 in acute myeloid leukemia (AML). Methods: We generated single-cell derived, biallelic LINC01547 knock-out clones in Kasumi-1 cells using the CRISPR/Cas9 system with paired sgRNAs. Knock-out and control cells were assessed for differences in proliferation and clonogenicity by cell growth and methylcellulose assays. Alterations in proliferation, cell cycle progression, apoptosis, and global protein synthesis were subsequently evaluated in flow cytometry-based assays. Finally, we characterized the impact of LINC01547 loss on RNA and protein homeostasis by RNA sequencing, as well as nascent and steady-state proteomics. Results: Bioinformatic analyses of expression data suggested a role for LINC01547 in AML. LINC01547 was highly expressed in AML (TCGA data), and its expression is associated with worse overall survival in AML. Biallelic deletion of LINC01547 suppressed AML cell growth and colony formation capacity. Similarly, EdU-based proliferation assays showed significantly reduced proliferation upon loss of LINC01547. Overall protein synthesis, assessed by O-propargyl-puromycin (OPP) assays, and apoptosis, analyzed by annexin V assays, were not significantly affected. Transcriptome and steady-state proteome analyses revealed that depletion of LINC01547 impacted the expression of multiple mRNAs and proteins. An overall weak correlation between differential expression on the transcript and the protein level indicated that LINC01547 might mediate its effects through both, transcriptional and post-transcriptional mechanisms. In line, mass-spectrometric analysis of nascent and steady-state proteomes upon LINC01547 depletion showed numerous alterations on the translational and total protein level, whereas the respective mRNAs often remained unaffected. Especially proteins involved in cell cycle regulation exhibited reduced translation rates upon loss of LINC01547. Consistently, cell cycle assays showed that depletion of LINC01547 slowed cell cycle progression and led to accumulation of cells in G0/G1 phase. Overlapping our transcriptome, total proteome, and nascent proteome data sets, we identify potential downstream targets through which LINC01547 presumably exerts its newly uncovered pro-oncogenic function. Summary/Conclusion: We demonstrate that the previously uncharacterized long non-coding RNA LINC01547 has wide-ranging functions in AML. Its overexpression in AML and negative correlation with overall survival are substantiated by its uncovered implications in cell proliferation, clonogenicity, and cell cycle progression. In-depth characterization and overlap of its impact on transcriptome, nascent proteome and total proteome reveal numerous genes and pathways affected by LINC01547 depletion. Further assessment of LINC01547 and its downstream targets will improve our understanding of the mechanisms underlying leukemogenesis and potentially reveal novel leverage points in AML therapy.